Because of the structural similarity between DNA and RNA, previous RNA purification methods have often comprised isolating DNA and RNA together from biological sources. One commonly used method for isolating nucleic acids from cells and tissues was the “Sevag” procedure. This method comprises contacting a cell or tissue homogenate with phenol or a mixture of phenol and chloroform, thereby denaturing proteins and precipitating them while leaving nucleic acids in solution. This method, while still used, is hazardous, laborious and of limited utility for isolation of RNA from biological sources containing high amounts of ribonuclease (RNase), an extremely stable enzyme that degrades RNA.
An improved method for isolating intact RNA from ribonuclease-rich tissues was disclosed by Chirgwin et al., Biochemistry, 18: 5924-29 (1979). This method comprises exposing tissue homogenates to concentrated guanidinium thiocyanate and 2-mercaptoethanol, thereby eliminating nucleolytic degradation of RNA by denaturing all of the cellular proteins, including ribonuclease, at a rate which exceeded the rate of RNA hydrolysis by ribonuclease. Although RNA isolated in this manner was biologically active, it was not free of contamination by DNA, protein and other cellular materials. Subsequent, often extensive, manipulation was required to further purify the RNA from other cellular contaminants.
Silica based nucleic acid isolation techniques have been developed as alternatives to, or in addition to, the conventional isolation techniques described above for use in isolating total RNA from at least some types of biological materials. For example, an RNA isolation kit has been developed that uses a glass fiber filter in a spin filter basket and a hybrid lysis buffer/binding solution with a high concentration of guanidine hydrochloride and a chaotropic agent to isolate total RNA from simple biological materials, such as cultured cells, blood, yeast, and bacteria (See, e.g. High Pure RNA Isolation Kit from Roche Diagnostics). A system for isolating total RNA from bacterial cells and tissue using a spin basket with a silica gel-based membrane, and a lysis buffer/binding solution containing guanidinium isothiocyanate has also been developed (See, e.g. the RNeasy total RNA kit from QIAGEN Inc, Chatsworth, Calif.). Both systems described briefly above allow one to isolate total RNA, but the yield and purity of RNA isolated tends to be low, particularly when used to isolate RNA from complex biological materials, such as plant or animal tissue.
As such, what is needed are methods, compositions, and kits that allow for the purification of RNA from complex biological materials with high yield, while avoiding DNA contamination.